The Active Components And Antioxidant Activity Of Fresh-cut Cistanche Deserticola Y. C. Ma By Modified Atmosphere Microporous MembranePackaging

Abstract: In order to study the postharvest storage quality of Cistanche deserticola planted in Xinjiang, the active modified atmospheretreatment (6% CO2+4% O2+90% N2) combined different packaging materials with PE film (oxygen permeation 300 cm3 /(m2·d)), microporousmembrane M1 (oxygen permeation 6 000 cm3 /(m2·d)) and microporous membrane M2 (oxygen permeation 8 000 cm3 /(m2·d)) were usedtotreatthe fresh-cut Cistanche deserticola. The effects on the changes of active components and antioxidant activities were studied under lowtemperature (4±0.5) ℃ storage. The results showed that PPO activity and browning degree in treatment group with modified atmospheremicroporous membrane(6% CO2+4% O2+90% N2+M1) were2.07 U·/g and 0.57 OD410/g, which were lower than CK group after storagefor7days. The contents of Vc, total phenols, flavonoids, total polysaccharides, echinoside and calycoside were 13.00%, 5.88%, 11.24%, 14.45%, 1.20% and 1.47% higher than those of CK group, respectively. In the meantime, the DPPH,ABTS + free radicalscavenging rate and FRAPvaluein 6% CO2+4% O2+90% N2+M1 microporous membrane treatment group were 8.97%, 1.99% and 11.43% higher than in CKgroup, respectively. In summary, 6% CO2+4% O2+90% N2+M1 treatment could significantly retard the decrease of active components, maintain higher antioxidant capacity and prolong the shelf life of C.deserticola. This study provides an efficient preservation method for fresh-cut C.deserticola whichbettermaintain the ability of medicine food homology.

Keywords: cistanche deserticola Y. C. Ma; modified atmosphere packaging; microporous membrane; inoxidizability

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Cistanche deserticola (Cistanche deserticola Y. C. Ma) is a parasitic plant of the genus Cistanche deserticola in the family Cistanche. It is warm in nature and sweet in taste. It contains various active substances such as polysaccharides, phenylethanol glycosides, flavonoids, polyphenols and alkaloids[1,2]. It has the functions of invigorating kidney yang, benefiting essence and blood, moistening intestines and laxative, relieving fatigue, delaying aging and

Enhance immunity and other effects [3,4]. At present, most of Cistanche sold in the market are dried products, and the traditional sun-dried method is used in the drying process, which causes the loss of some active ingredients in Cistanche and weakens the efficacy of Cistanche. Fresh-cut fruits and vegetables have the characteristics of convenience, quickness and high degree of freshness. They are deeply loved by consumers and have gradually become the mainstream of fresh fruits and vegetables processing [5]. Modified atmosphere packaging is widely used in fruit and vegetable preservation due to its high efficiency, safety, and low cost. Microenvironment-modified atmosphere treatment effectively slowed down the decline of total soluble solids (TSS), titratable acid (TA), Vc and anthocyanin contents of blueberry fruit during storage, so that it still maintained a high nutritional value[6] . Controlled atmosphere combined with phase temperature treatment can effectively maintain the content of reducing sugar, soluble protein and flavonoids in lily, inhibit the formation of alcohols and esters, improve antioxidant capacity, and reduce the occurrence of browning [7].

The microporous membrane combines its own specific air permeability with the respiration of fruits and vegetables to spontaneously adjust the gas composition in the package [8], so that the gas ratio in the package reaches a dynamic balance, effectively delaying the decline in the storage quality and oxidative aging of fruits and vegetables [9] . Microporous membrane modified atmosphere packaging can effectively slow down the decline of soluble protein content and chlorophyll content of green edamame [10], effectively reduce the degradation of chlorophyll in cucumber, slow down the production of O2-, and enhance the activity of related antioxidant enzymes at the same time. Stress resistance of cucumber [11].

Increased the content of total phenols and total anthocyanins in pomegranate peel, and enhanced the antioxidant activity [12]. There are few reports on the modified atmosphere microporous membrane packaging technology in the research of fresh-cut Cistanche deserticola. Modified atmosphere microporous membrane packaging can effectively maintain the nutrients in fruits and vegetables, and has a significant impact on the antioxidant capacity [11,13], but there are few studies on the changes in the active components and antioxidant properties of fresh-cut Cistanche deserticola. Therefore, in this paper, the modified atmosphere microporous membrane was used to package the fresh-cut Cistanche, and the changes of the active components of the fresh-cut Cistanche and their effects on the antioxidant activity during storage were studied. In order to provide a technical basis for the study on the homology of medicine and food of Cistanche deserticola.

1 Materials and methods

1.1 Materials and reagents

Cistanche: purchased in Hotan, Xinjiang in November 2021 and transported to a cold storage for 24 hours at 10°C. Fresh Cistanche with no mechanical damage, no pests and diseases, and uniform size and thickness (about 4 cm in diameter) was selected for subsequent experimental research. PE film (thickness 40 μm, oxygen permeability 300 cm3/(m2 d)), 6 000-pore microporous membrane (thickness 25 μm, oxygen permeability 6 000 cm3

/(m2 d)), 8 000-pore microporous membrane (thickness 25 μm, oxygen permeability 8 000 cm3/(m2 d)), all provided by Jiangsu Jiubang New Material Technology Development Co., Ltd. Chromatographically pure acetonitrile and formic acid, Merck, Germany; standard chromatographically pure verbascoside and echinacoside, Abel Co., Ltd.; sodium chloride, citric acid, sodium bisulfite, L-cysteine, calcium chloride, Sodium hypochlorite, guaiacol, polyethylene glycol, catechol, ascorbic acid, potassium persulfate (K2S2O8) Tianjin Guangfu Fine Chemical Research Institute; 1,1-diphenyl-2-trinitrophenylhydrazine (1 ,1-diphenyl-2-picrylhdrazyl, DPPH), 2,2'-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (2,2'-azino-bis(3 -ethylbenzothiazoline-6)-sulphonic aciddiammonium salt), ABTS), 2,4,6-tripyridyltriazine (2,4,6-Tris(2-pyridyl)-s-triazine, TPTZ), Beijing Cool Chemical Technology Ltd.; the above reagents are of analytical grade.

1.2 Test equipment

UV-2600 ultraviolet spectrophotometer, Shimadzu Corporation, Japan; HC-3018R high-speed refrigerated centrifuge, Agilent-1100 high-performance liquid chromatography, PerkinElmer, USA; MS105DU 1/100,000 analytical balance, Mettler Toledo, Switzerland ; SPX-100B-Z constant temperature and humidity box, Shanghai Boxun Industrial Co., Ltd.

1.3 Test method

Fresh cistanche after precooling for 24 hours was peeled, washed, cut into pieces, color-protected and sterilized, and then put into packing boxes (length×width×height=180 mm×140 mm×5 mm, 200 g per box). PE film, microporous film with 6 000 pores, and microporous film with 8 000 pores were used for modified atmosphere packaging (heat-sealing temperature was 140 °C, heat-sealing time was 2 s, and the ratio of modified atmosphere was 4% O2+6% CO2+ 90% N2), which are denoted as CK, M1 and M2 in the text, respectively. Immediately after treatment, they were stored in a constant temperature incubator at a temperature of (4±0.5)°C and a relative humidity of (90±1)%. Each treatment was repeated 3 times, and samples were taken every 1 day for a total of 7 days. After the samples were pulverized, they were treated with liquid nitrogen and stored in a -40 °C refrigerator for the determination of subsequent indicators.

1.4 Index determination method

1.4.1 Determination of O2, CO2 volume fraction, PPO activity and browning degree

The Check point 3 portable headspace analyzer was used to regularly measure the percentages of O2 and CO2 in the packaging of different treatment groups, the unit is %, and the determination of the activity of each PPO was carried out according to the method of Cao Jiankang [14].

The degree of browning was determined by the extinction value method [14] with a slight modification. Accurately weigh 2.0 g of Cistanche cistanche, put it into a 50 mL centrifuge tube after homogenization, add distilled water at a ratio of 1:10 (g:mL), centrifuge at 4 °C, 10 000 × g for 5 min, take the supernatant and put it in a 25 °C water bath Soak in the pot at constant temperature for 5 min, measure the absorbance of the supernatant at 410 nm, and express the result as OD410/g.

1.4.2 Determination of Vc, total phenols and flavonoids

Determination of Vc content, total phenolic content and flavonoid content: using spectrophotometric method [14].

1.4.3 Determination of total polysaccharide content

It was determined by the phenol-sulfuric acid method, and slightly modified according to the method of Zhao Yan et al. [15]. Preparation of sample liquid: Accurately weigh 1.0 g of Cistanche cistanche sample powder, according to the ratio of solid to liquid 1:30 (deionized water), ultrasonically extract at 50 °C for 60 min, centrifuge at 4 °C, 8000×g for 5 min, and take supernatant, add

Add 95% ethanol until the ethanol concentration is 80%, let stand at 4 °C for 12 h, discard the supernatant, wash the precipitate with absolute ethanol and acetone twice, add deionized water, and use sevage solution ( Chloroform: n-butanol = 4:1) to remove the protein, and to be tested after constant volume. Add 600 μL of 6% phenol solution and 3 mL of concentrated sulfuric acid to 1 mL of the sample solution, mix and place in a boiling water bath for 10 min, and measure the absorbance at 490 nm after cooling. Prepare the standard solution with glucose to draw the standard curve equation, and the measurement results are expressed in glucose equivalent (mg DE/g DW).

1.4.4 Determination of echinacoside and verbascoside

Preparation of reference substances: Take appropriate amount of standard substances of verbascoside and echinacoside (both purity ≥ 98%), measure them accurately respectively, add 50% methanol to make a stock solution with a concentration of 1.0 mg/mL, and then take an appropriate amount of stock solution solution mixed to obtain the respective concentration of 0.05 mg/mL, 0.10 mg/mL, 0.15 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL

mixture. Take the peak area (Y) as the ordinate, and draw the standard curve with the quality of the reference substance (X, mg).

Preparation of the test solution: vacuum freeze-dry the sample frozen in liquid nitrogen, pass through (No. 4) sieve after freeze-drying. Precisely weigh 1.0 g of cistanche powder, put it in a 50 mL brown measuring bottle, add 25 mL of 50% methanol, shake well and soak for 30 min, sonicate for 40 min, cool

Then add 50% methanol to the weight before ultrasonication, let it stand still, take the supernatant, and filter it with a 0.45 μm microporous membrane.

Chromatographic conditions: Agilent Eclipse XDB-C18 column (4.6 mm×250 mm, 5 μm), detection wavelength 254 nm), column temperature 25 ℃; acetonitrile (A)-0.1% formic acid aqueous solution (B) as flow Phase, gradient elution (0-20 min, 5%-15% A; 20-40 min, 15%-30%); flow rate 1.0 mL/min, injection volume 10 μL.

1.4.5 Determination of antioxidant activity in vitro

1.4.5.1 DPPH free radical scavenging ability[16]

Precisely prepare 0.2 mmol/L DPPH ethanol solution, and place it under dark conditions (ready for immediate use). Ai: 0.5 mL 0.2 mmol/LDPPH ethanol solution; Ac: 0.5 mL absolute ethanol + 0.5 mL 0.2 mmol/LDPPH ethanol solution; Aj: 0.5 mL sample solution + 0.5 mL absolute ethanol. Store in the dark at room temperature for 30 minutes, measure the absorbance value at 517 nm, and calculate according to the following formula: DPPH free radical scavenging rate/% = [1Ai-AjAc ] × 100 (1)

1.4.5.2 Determination of ABTS+ free radical scavenging ability refers to the method of Tang Yanping [17].

1.4.5.3 The determination of ferric reducing/antioxidantpower (FRAP) is determined according to the method of Wang Miaomiao et al. [18].

1.5 Data statistics and analysis

Excel 2010 was used for data processing, SPSS20.0 was used for one-way analysis of variance, and GraphPad Prism8.0 software was used for graphing. P≤0.05 indicated significant difference, and ≤0.01 indicated extremely significant difference.